Composite neural conduit

ABSTRACT

A composite nerve conduit comprising an elongated body comprising one or more hollow elongated internal channels for guiding and promoting nerve regeneration. The conduit is a three-dimensional scaffold comprising a crosslinked hybrid/composite matrix of collagen and soy protein isolate having improved mechanical and biocompatibility properties. Methods of using the conduit for promoting nerve regeneration at a site of neural tissue damage by bridging wounded, severed, or damaged nerve sections in a peripheral and/or central nervous system. Methods of fabricating composite neural conduits are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the priority benefit of U.S. ProvisionalPatent Application Ser. No. 62/648,208, filed Mar. 26, 2018, entitledCOMPOSITE NEURAL CONDUIT, incorporated by reference in its entiretyherein.

BACKGROUND Field of the Invention

The present invention relates to composite neural conduits havingimproved properties for repair of neural defects and damage.

Description of Related Art

In the field of neural regeneration, neural conduits are used to repairneural defects. Conduits act like a bridge and connect two damaged nerveendings together, providing a channel and scaffold to guide andfacilitate nerve growth. As the new nerves grow along the conduit, thetwo ends can reconnect and restore function. Restoration of function inthe peripheral nervous system is possible, but there are a number offactors surrounding the repair of the central nervous system to make itmuch more challenging.

A variety of conduit designs and compositions have been explored, butsuccess in clinical applications find some materials and configurationsare advantageous compared to others. For example, bovine collagen is acommon biomaterial that mimics a microenvironment suitable for neuralgrowth, but there are issues with mechanical defects and inflammatoryresponses occurring.

SUMMARY OF THE INVENTION

The present disclosure is concerned with composite nerve conduitscomprising a three-dimensional crosslinked composite matrix of collagenand soy protein isolate configured with one or more hollow elongatedinternal channels for guiding and promoting nerve regeneration. Theneural conduits are fabricated as elongated bodies configured tosubstantially encircle at least two damaged neural sections in aperipheral and/or central nervous system and bridge any gaptherebetween. The matrix material used to form the conduit can consistessentially or even consist of crosslinked collagen and soy proteinisolate (including any remaining crosslinker, if applicable).

Methods of promoting nerve regeneration at a site of neural tissuedamage by bridging wounded, severed, or damaged nerve sections in aperipheral and/or central nervous system are also described herein. Themethods generally comprise one damaged nerve end with a first end of acomposite neural conduit according to the various described embodiments,and contacting a second end of the neural conduit with an opposingdamaged nerve end, to thus bridge the gap between the damaged sections.In some embodiments, treatment modalities also include applying anelectric current to promote regeneration.

The present disclosure also concerns methods of fabricating compositeneural conduits according to the various described embodiments. Themethods generally comprise providing a biopolymer solution comprisingcollagen and soy protein isolate dissolved or dispersed in a solventsystem. The biopolymer solution is applied to a negative mold to formthe body of the conduit. The negative mold is generally configured withone or more elongated removable inserts for forming one or more hollowelongated internal channels within the body. The biopolymer solution isthen solidified in the presence of the mold to yield a crosslinkedmatrix comprising a composite of collagen and soy protein isolate. Thecrosslinked matrix is then removed from the mold (or vice versa, themold is removed from the matrix) to yield an elongated body comprisingthe one or more hollow elongated internal channels extending betweenrespective terminal ends of the body.

The composite/hybrid neural conduits have improved properties overconventional conduit materials, including stronger structural integrity,resiliency, and improved biocompatibility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a plan view of a composite neural conduit having multiplechannels in accordance with an embodiment of the invention;

FIG. 1B is a cross-sectional view of the composite neural conduit inFIG. 1A;

FIG. 2 shows a photographic image of the SPI-collagen composite4-channel conduit fabricated in Example 2 and compared with atraditional collagen conduit (left panel), and a photographic image of across-section of the SPI-collagen composite 4-channel conduit (rightpanel);

FIG. 3 is a photographic image of the bending test to show theflexibility and resiliency of the SPI-collagen composite 4-channelconduit (a) before, (b) during, and (c) after manually bending a lengthof the conduit tube in half;

FIG. 4 is a graph showing the data from the compressive test of neuralconduits showing the (a) flexibility/resiliency; and (b) stiffness ofthe conduits;

FIG. 5 is a graph showing the data from the FTIR test comparingSPI-collagen composite and collagen conduits;

FIG. 6 is a graph of the data for the degradation study of SPI-collagencomposite conduits and collagen conduits;

FIG. 7 is a graph of the data for the swelling test of the conduits,showing changes in (a) width and (b) length of a given fabricatedconduit; and

FIG. 8 shows images of neural cell growth on A) collagen and B)SPI-collagen composite, in which the cells are labeled with anti-βIIItubulin antibody.

DETAILED DESCRIPTION

It is an object of the present invention to provide a neural conduitsuitable for the repair of nerves of the peripheral and/or centralnervous systems in a patient. Another object of the invention is toprovide a biomaterial scaffold with favorable material and mechanicalproperties. A further object is to provide a multichannel compositenerve conduit with improved physical and biomechanical properties. Thecomposite nerve conduits are biocompatible and biodegradable, andpromote the growth of nerves in a regular and controlled manner. Anotherobject is to provide a flexible, but strong nerve conduit material, withdecreased inflammatory effects. Composite neural conduits of theinvention have greater structural integrity and stiffness as compared toconventional collagen conduits, but retain suitable flexibility andresiliency such that the conduit can flex and bend, but will spring backinto its original form after bending. In one or more embodiments, thecomposite neural conduits have a stiffness (N/mm) of greater than about6.5, preferably greater than about 7, more preferably greater than about8. The soy protein isolate in the conduit can be used to modulate thestructural and mechanical properties of the fabricated conduits.Further, the composite conduits retain their original form better overtime in vivo as compared to conventional collagen conduits, and degrademore slowly than conventional collagen conduits with reduced swelling.Further, the composite neural conduits have improved biocompatibility ascompared to conventional collagen conduits, as demonstrated by increasedneural outgrowth.

The presented technology is a multi-channel neural conduit with a hybridcomposition comprising a mixture of collagen and soy protein isolate asthe structural polymer matrix. Soy protein isolate imparts favorablebioactivity, biodegradability, biocompatibility, and processability tothe conduit material. Utilizing a hybrid collagen-soy protein isolateconduit results in improved mechanical and biological properties,increasing the ability and likelihood for a nerve defect to be repaired.Advantageously, soy protein isolate reduces immunological andinflammatory responses, while increasing mechanical strength.

The hybrid/composite material comprises an unexpected mixture of animaland plant proteins to promote nerve regeneration, bridging wounded,severed, or damaged nerve sections and allowing the nerve to re-growalong the composite channel(s).

The composite nerve conduit is formed by preparing a biopolymer solutioncomprising (consisting essentially or consisting of) collagen and soyprotein isolate dissolved or dispersed in a solvent system. Thebiopolymer solution is prepared by mixing collagen and soy proteinisolate in a suitable solvent system. For example, separate solutions ofcollage and soy protein isolate can be first prepared, followed bymixing the solutions in the desired ratios. Alternative, collagen powerand/or soy protein isolate powder can be directly mixed into a suitablesolvent system. Exemplary solvent systems include glycerol, acetic acid,water, and mixture thereof.

Various weight ratios of collagen and soy protein isolate may be used,depending upon the desired properties of the final matrix. In one ormore embodiments, the weight ratio of collagen to soy protein isolate inthe biopolymer solution is about from about 20:80 to about 80:20, morepreferably from about 30:70 to about 70:30, more preferably from about40:60 to about 60:40, and even more preferably about 50:50.

Preferably, the collagen is type I collagen, and more preferablycollagen extracted from bovine tendon using standard protocols. Thecollagen is generally provided in powder form before mixing with thesolvent system as described. Preferably, the soy protein isolate is 92%SPI (Soy Protein Isolate, MP Biomedicals™). It is appreciated that soyprotein isolate contains high levels of genistein, one of the principalisoflavones. In other research, genistein has been shown to act as akinase inhibitor and has demonstrated immunosuppressive andanti-inflammatory functions. Soy protein isolate provides additionalbenefits of decreasing the level of proinflammatory cytokine productionof mononuclear cells from animal peripheral blood and therefore controlimmunological reaction. Isoflavones, like genistein, can further improvetissue regeneration and wound healing through an estrogenreceptor-independent mechanism. Meanwhile, the soy protein moleculeitself has tunable structures and may generate the desirable mechanicalproperties depending on the processing method. Soy protein isolate maybe fabricated into a variety of biomaterial structures including neuralconduits and hydrogels. Preferably the soy protein isolate is generallyprovided in powder form before mixing with the solvent system asdescribed.

The resulting biopolymer solution (comprising or consisting of a mixtureof collagen and soy protein isolate) is then molded into the desiredgeometry for the neural conduit body. Molding may be achieved, forexample, by extrusion, injection molding, blow molding, and the like.The viscosity of the biopolymer solution can be adjusted using more orless solvent for the appropriate molding technique. The % by weight ofbiopolymer in the solution will generally range from about 0.01 to about20% weight, based upon the total volume of the solution taken as 100%.

In one or more embodiments, the neural conduit body is formed byapplying the biopolymer solution around a removable insert that servesas the “negative” for the neural conduit form, such as an elongated wireor tube (e.g., stainless steel wire, plastic, etc.). The negative moldcan include a plurality of elongated wires for forming a plurality ofinternal elongated hollow channels within the neural conduit body whenthe removable insert is subsequently removed. The biopolymer solution isthen solidified around the negative mold.

In one or more embodiments, the biopolymer solution is first air driedto remove residual solvent. In one or more embodiments, the biopolymersolution is then crosslinked. This can be accomplished by addition ofone or more crosslinking agents to the biopolymer solution. Exemplarycrosslinking agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), transglutaminase,glutaraldehyde, sulfonates, genipin, and the like. After crosslinking,the resulting crosslinked matrix should be washed to remove residualcrosslinker, e.g., with distilled water and/or monosodium phosphate.After washing, the crosslinked matrix can be lyophilized, followed byremoval of the crosslinked matrix from the negative mold. It will beappreciated that alternative crosslinking mechanisms may be useddepending upon functionalization of the biopolymers, including thermalcrosslinking and photo crosslinking.

The resulting composite neural conduit comprising an elongated body(e.g., single or multi-channel tube) configured for implantation at asite of neural tissue damage for guiding axon regeneration and neuralcell migration in order to establish new functional connection acrossthe lesion. The composite neural conduit provides a three-dimensionalscaffold for bridging a nerve gap or defect. With reference to FIGS. 1Aand 1B, the resulting neural conduit body 10 comprises one or morehollow channels 12 for guiding and promoting nerve regeneration, formedout of the crosslinked composite crosslinked composite matrix 14 ofcollagen and soy protein isolate and defined by respective interiorsidewall surfaces 14′. In general, the body 10 is elongated and extendsbetween two respective terminal ends 10 a, 10 b so as to fit around orbridge a gap between the two damaged segments of the injured nerve inthe patient (not shown). The body 10 presents an external surface 16 andcan be cylindrical in shape, such that the radial exterior surface 16presents a substantially circular axial cross section. However, othergeometric shapes are contemplated (e.g., oval, elliptical, polygonal,such as octagonal, etc.) without departing from the teachings of thepresent invention. The internal channels 12 can likewise be cylindricalwith a radial interior sidewall 14′ that likewise presents asubstantially circular axial cross-section; however, it will beappreciated that the cross-sectional shape will depend upon the shape ofthe negative form used in molding the channels 12. Thus, it will bereadily apparent to one of ordinary skill in the art that the term“radial,” as used herein with respect to the conduit, is not limited tosubstantially circular cross sections and encompasses polygonal crosssections or cross sections presenting other geometric shapes (e.g., ovalor elliptical cross sections).

The composite neural conduit matrix that defines the conduit body formis of a resilient, biocompatible material, and the body comprises afirst end 10 a for connecting to a first end of the damaged nerve, and asecond end 10 b for connecting to the second end of an opposing damagednerve. The internal hollow channels 12 of the body 10 extend between thefirst and second terminal ends 10 a, 10 b of the conduit body 10 tofacilitate rejoining of the damaged nerve ends. The nerve conduit mayhave a plurality of channels 12, such as 1 or more, preferably 2 ormore, more preferably from 2 to 10, more preferably 2 to 7, and evenmore preferably from 2 to 4 channels.

Preferably, the channels are evenly sized and spaced apart. This isachieved by evenly spacing the elongated negative mold forms, and usingmold forms of the same size. The neural conduit cross-sectionaldimension (e.g., diameter, maximum width, etc.), circumference, length,and channel diameter will vary depending on the species of the patient,size of the nerve, area of injury, and extent of injury to be repaired.A variety of dimensions can be used. Non-limiting examples are describedherein. The outer diameter (cross-sectional dimension or width) asmeasured from the exterior side wall 16 of the nerve conduit body can beup to 4 cm, with the diameter of each channel 12 ranging between about50 μm and about 4 mm. For a 2-mm diameter conduit, the diameter ofrespective channels may be about 50 μm to about 700 μm, preferably about530 μm. It will be appreciated that the channel number and diameter canvary widely, especially depending upon the particular patient. For humanpatients, the conduit may vary from 1 channel to 100 channels, and thechannel diameter can range from about 10 micrometers to 2 cm. In use,the conduit can be cut-to-size, and short lengths of the conduits aregenerally used, with typical manufactured lengths being 100 mm or less,preferably 75 mm or less, and in some cases 50 mm or less, 30 mm orless, 15 mm or less, 10 mm or less, or 5 mm or less. General dimensionsrange from 1-2 mm in diameter up to 4-5 mm in diameter, and 5 mm inlength up to 100 mm in length for nerves and up to 4 cm in diameter forspinal cord repairs (with lengths ranging from 5 mm to 100 mm).

The term “diameter” is used herein for ease of reference to refer to thelargest cross-sectional dimension, aka width, and should not beconstrued as excluding similarly measured dimensions for conduitgeometries that are not circular in their cross-sectional shape. It isfurther noted that the dimensions of the illustrated body 10 areprovided by way of example only and are not to be construed as limiting,as numerous shapes and/or sizes of bodies may be alternativelyconfigured, as will be readily appreciated by one of ordinary skill inthe art upon review of this disclosure. Moreover, while the illustratedembodiment includes the plurality of channels 12 being arranged in aconfiguration of generally equally spaced groupings, such a normalpattern is by way of example only, and is not necessarily required. Forinstance, the plurality of channels 12 could alternatively be configuredin a generally uniform staggered arrangement or an entirely randomarrangement, without departing from the teachings of the presentinvention.

In one or more embodiments, the neural conduit further comprises one ormore materials filling the internal hollow channels 12 of the body 10,such as neurotrophic materials, anti-inflammatories, therapeutic agents,antibiotics, or other active agents, for example to support cellulargrowth and neural regeneration. In one or more embodiments, the neuralconduit is filled with hydrogel and/or aligned nanofibers. For purposesof the present disclosure, hydrogel refers to a solid jelly-likematerial where the liquid component is water. The hydrogel is preferablya network of polymer chains that are hydrophilic, where water is thedispersion medium. The hydrogel is preferably highly absorbent andpossess a degree of flexibility very similar to natural neural tissue.The hydrogel preferably comprises at least one component selected from,but not limited to, a collagen/soy protein composite hydrogel (seeExample 3), chitin, chitosan, guar gum, gum karaya, agar, treated agar,fenugreek seed mucilage, soy polysaccharide, gellan gum, mango peelpectin, lepidium sativum mucilage, plantago ovata seed mucilage, aeglemarmelos gum (AMG), locust bean gum, ficus indica fruit mucilage,Mangifera indica gum (MIG), hibiscus rosa sinesis mucilage and treatedagar, dehydrated banana powder (DBP), collagen, fibrin, fibronectin,laminin, hyaluronic acid, polysilozane, polyphosphazene, low-densitypolyethylene (LDPE), high-density polyethylene (HDPE), plastic,polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), nylon,nylon 6, nylon 66, Teflon (polytetrafluoroethylene), thermoplasticpolyurethanes (TPU), polylacticoglycolic acid (PLGA), polycaprolactone(PCL), and combinations thereof.

In one aspect, the hydrogel comprises further components in or added tothe hydrogel matrix within the channels 12. These further components,include, but are not limited to, neural cells, stem cells, growthfactors, proteins, progenitor cells, therapeutic peptides, gene vectors,siRNA, miRNA, chemical compounds, and combinations thereof. In one ormore embodiments, the composite neural conduit is essentially free ofany other additives or components, except those expressly describedherein, such as coatings, metals, support structures, and the like. Theterm “essentially free” means that such component is not intentionallyadded (although residual amounts or impurities of the component may bepresent).

Also contemplated herein are methods of promoting nerve regeneration inperipheral and/or central nervous systems, using the composite neuralconduits. The methods comprise connecting a first end of a compositeneural conduit according to the invention with one end, either rostralor caudal stump, of the damaged spinal cord or nerve. The second end ofthe composite neural conduit is connected with an opposing damaged nerveend. The neural conduit can be secured in place using any suitablebiocompatible technique, including suturing. Such attachment means maybe a perforation down the length of the neural conduit so that it may bewrapped around the area where the neural cell or nervous tissue has beeninjured, and sutured into place if desired. Thus, in the method, thecomposite neural conduit is generally implanted at a “site of nervetissue damage,” which refers to the damaged segment itself, as well as(relatively) undamaged adjacent segments or stumps on either side of thedamaged segment (or gap). The type of nerve injury may be any damage toneural cells or nervous tissue within the body of the patient. Thedamaged segment may be compression, lesion, tear, or the like, or mayinclude a completely transected nerve. The nerve injury can occur incentral nervous system (CNS) or the peripheral nervous system (PNS).Injuries of the CNS may occur anywhere in the CNS, such as, but notlimited to the spinal cord or brain. Injuries of the PNS may occuranywhere in the PNS, such as body extremities, but not limited to, face,arms, hands, legs, feet, or phalanges. In a preferred embodiment, thenerve injury is preferably selected from, but not limited to spinal cordinjury, nerve injury, neuropraxia, axonotmesis, and neurotmesis.

Additional methods of promoting nerve regeneration may include theapplication of electrical stimulation to a damaged nerve bridged by thecomposite neural conduit. The application of electrical stimulation to adamaged nerve segment may act as a therapeutic regimen for promotingunidirectional cell migration and axonal growth through a neuralconduit. In one or more embodiments, electrical stimulation may bedelivered to the damaged nerve site by an electrode comprising an anodalelectrode and cathodal electrode. In one or more embodiments, the anodalelectrode may be applied to a rostral stump of damaged nerve (i.e.,upstream of the injured or damaged portion). In one or more embodiments,the cathodal electrode may be applied to a composite neural conduit thatis bridging the defect, injury, or lesion in the damaged nerve.Exemplary approaches for electrical stimulation are described in Int'lPub. No. WO/2018/232145 (PCT/US2018/037585), filed Jun. 14, 2018,incorporated by reference herein in its entirety.

The patient for purposes of this disclosure may be any human or animalhaving a neural injury. Non-limiting examples include, human andnon-human mammals, such as dogs, cats, equine, bovine, or porcinesubjects, goats, rodents (e.g., rats, rabbits, mice), elephants,monkeys, gorillas, zebras, camels, lions, tigers, bears, and the like.

Additional advantages of the various embodiments of the invention willbe apparent to those skilled in the art upon review of the disclosureherein and the working examples below. It will be appreciated that thevarious embodiments described herein are not necessarily mutuallyexclusive unless otherwise indicated herein. For example, a featuredescribed or depicted in one embodiment may also be included in otherembodiments, but is not necessarily included. Thus, the presentinvention encompasses a variety of combinations and/or integrations ofthe specific embodiments described herein.

As used herein, the phrase “and/or,” when used in a list of two or moreitems, means that any one of the listed items can be employed by itselfor any combination of two or more of the listed items can be employed.For example, if a composition is described as containing or excludingcomponents A, B, and/or C, the composition can contain or exclude Aalone; B alone; C alone; A and B in combination; A and C in combination;B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certainparameters relating to various embodiments of the invention. It shouldbe understood that when numerical ranges are provided, such ranges areto be construed as providing literal support for claim limitations thatonly recite the lower value of the range as well as claim limitationsthat only recite the upper value of the range. For example, a disclosednumerical range of about 10 to about 100 provides literal support for aclaim reciting “greater than about 10” (with no upper bounds) and aclaim reciting “less than about 100” (with no lower bounds).

The present invention is susceptible of embodiment in many differentforms. While the drawings illustrate, and the specification describes,certain preferred embodiments of the invention, it is to be understoodthat such disclosure is by way of example only. There is no intent tolimit the principles of the present invention to the particulardisclosed embodiments.

EXAMPLES

The following examples set forth methods in accordance with theinvention. It is to be understood, however, that these examples areprovided by way of illustration and nothing therein should be taken as alimitation upon the overall scope of the invention.

Example 1—General Protocol Fabrication of Multichannel Collagen-SoybeanNeural Conduits

Soybean protein isolate (SPI) solutions will be prepared by slowlydissolving the SPI with constant stirring in distilled water with theconcentration of 3-15% w/v. The pH may be adjusted to 7.2 with 1M sodiumhydroxide or 1M HCl. Then glycerol, 50% w/w, (relative to SPI) will beadded to the solution with constant stirring, which helps to stabilizethe SPI in solution. The solution will be heated at a constanttemperature of 55° C. for 30 min and cooled at room temperature. Thenthe soybean solution will be mixed with type I collagen solution (10mg/ml, in acetic acid) as different SPI to collagen ratios (weight toweight) to fabricate neural conduits. The neural conduits are fabricatedusing the type I collagen-SPI composite according to fabrication methodsdescribed in U.S. Pat. No. 8,926,886, incorporated by reference in itsentirety herein. Briefly, collagen-SPI solution sequentiallyself-assembles on the molds with insertion of stainless-steel wires.Then the conduit will be crosslinked with EDC and NHS. After washingwith NaH₂PO₄ (0.1 M) and distilled water, the composite is freeze-driedon the wires. Molds and wires will be removed from the neural conduitsafter freeze-drying.

Example 2—Composite Neural Conduit 1. Fabrication and MechanicalProperties 1.1 Fabrication of Multichannel Collagen-Soybean NeuralConduits.

Soybean protein isolate (SPI) solutions were prepared by slowlydissolving the SPI (Soy Protein Isolate, MP Biomedicals™) with constantstirring in distilled water with the concentration of 10% w/v. Thenglycerol, 50% w/w, (relative to SPI) was added to the solution withconstant stirring. The solution was heated at a constant temperature of55° C. for 30 min and cooled at room temperature. Then the soybeansolution was mixed with type I collagen solution (isolated in lab frombovine tendon; 10 mg/ml, in 50 mM acetic acid) as 1:1 protein weightratio to fabricate neural conduits. Then 4-channel conduits werefabricated. The matrix was crosslinked with 30 m EDC and 20 mM NHS.After washing with NaH₂PO₄ (0.1 M) and distilled water, the collagen wasfreeze-dried on the wires. The resulting conduits are shown in FIG. 2.

1.2 Mechanical Property of the Neural Conduits.

Mechanical properties of multichannel conduits were characterized bycompressive tests. The compressive tests were performed on thefabricated neural conduits using a uniaxial test machine (ELF 3200Endura-Tec, Schaumburg, Ill.). FIGS. 3 and 4 show that the stiffness ofSPI-collagen is higher than the collagen conduits, and its resiliency isimproved.

2. Chemical Properties 2.1 Fourier Transform Infrared Spectroscopy(FTIR) Assay.

Fourier transform infrared spectroscopy (FTIR) was performed to studythe chemical bond formation of collagen-SPI hydrogels with or withoutcrosslinking. Spectra was obtained using a Spectrum 100 FT-IRSpectrometer Perkin Elmer (PerkinElmer). FIG. 5 confirms thecrosslinking of the conduits by EDC/NHS.

2.2 Degradation Test.

Bacterial collagenase was used to study the degradation profile ofcollagen conduits. The enzyme was dissolved in a 0.1 M Tris-HCl buffer(pH 7.4) solution containing 0.005 M CaCl₂. After 1-day and 3-dayincubation of the samples with the enzyme solution, the remaining pelletwere repeatedly washed with distilled water and then freeze-dried. Thesamples were then be weighed. FIG. 6 shows that SPI-collagen conduitsand collagen conduits are biodegradable by collagenase.

2.3 Conduit Swelling Test.

The neural conduits were placed in PBS (pH 7.4). The length and width ofthe conduits were studied at time points of immediately beforeincubation and at day 1, 7 and 30 after incubation. The length and widthof conduits were measured with a digital caliper. The results are shownin FIG. 7, panels (a) and (b).

3. Biocompatibility

3.1 the Study of Neurite Outgrowth from Human-Induced Pluripotent StemCell-Derived Neurons on Collagen-Soybean Composite Films.

Collagen films were fabricated by spreading the collagen-SPI solution ona flat Teflon surface (weigh boat) and air-dried. The collagen filmswere then crosslinked with EDC and NHS as above. The induced pluripotentstem cell-derived neurons were grown on the films. The study showed theextensive neurite growth on SPI-collagen films. The study indicates thatthe crosslinked SPI-collagen films are biocompatible, and have improvedbiocompatibility as compared to collagen, as shown in the images in FIG.8.

Example 3—Composite Neural Hydrogel 1. Fabrication and MechanicalProperties 1.1 Fabrication of a Composite Collagen-Soybean Hydrogels.

SPI solutions are prepared by adding SPI powder in distilled water withthe concentration of 1-10% w/v. Then, glycerol is added to the SPIsolution under mechanical stirring at a constant temperature of 55° C.for 30 min and cooled at room temperature.

The SPI solution is mixed with type I collagen solution (in acetic acid)at various weight ratios, respectively, to form an SPI/collagensolution. To form the hydrogel, the pH value of the SPI/collagensolution is adjusted to 7 by adding a NaOH aqueous solution (1M) andphosphate-buffered saline (PBS) solution (10λ). The gelation takes placeby incubating the SPI/collagen solution in the 37° C. incubator.

The mixture of SPI-collagen solution is crosslinked with 4S-StarPEG atvarious concentrations. The final 4S-StarPEG concentrations in theSPI-collagen will range between 0.05 mM and 8 mM. A collagen hydrogellacking crosslinker will be used as a control.

1. A composite nerve conduit comprising an elongated body comprising oneor more hollow elongated internal channels for guiding and promotingnerve regeneration, said one or more hollow elongated internal channelsdefined by a sidewall comprising a crosslinked composite matrix ofcollagen and soy protein isolate, said body having respective first andsecond terminal ends, wherein said one or more internal channels extendsbetween the first and second ends of the conduit body.
 2. The compositenerve conduit of claim 1, comprising two or more of said hollowelongated internal channels for guiding and promoting nerveregeneration.
 3. The composite nerve conduit of claim 1, said bodyhaving cross-sectional dimension of from about 1 mm to about 4 cm and alength of from about 5 mm to about 100 mm.
 4. The composite nerveconduit of claim 1, wherein the neural conduit is configured tosubstantially encircle at least two damaged neural sections in aperipheral and/or central nervous system and bridge any gaptherebetween.
 5. The composite nerve conduit of claim 1, wherein saidneural conduit comprises one or more additional elements selected fromthe group consisting of growth factors, stem cells, neural cells,progenitor cells, gene vectors, proteins, therapeutic peptides, siRNA,miRNA, chemical compounds, and combinations thereof adsorbed thereon ortherein.
 6. The composite nerve conduit of claim 1, said one or morehollow elongated internal channels is filled with a hydrogel comprisingat least one material selected from the group consisting of acollagen/soy protein composite, chitin, chitosan, guar gum, gum karaya,agar, treated agar, fenugreek seed mucilage, soy polysaccharide, gellangum, mango peel pectin, lepidium sativum mucilage, plantago ovata seedmucilage, aegle marmelos gum, locust bean gum, ficus indica fruitmucilage, Mangifera indica gum, hibiscus rosa sinesis mucilage andtreated agar, dehydrated banana powder, collagen, fibrin, fibronectin,laminin, hyaluronic acid, polysilozane, polyphosphazene, low-densitypolyethylene, high-density polyethylene, plastic, polypropylene,polyvinyl chloride, polystyrene, nylon, nylon-6, nylon-66, Teflonthermoplastic polyurethanes, polylacticoglycolic acid, polycaprolactone,and combinations thereof.
 7. The composite nerve conduit of claim 1,said sidewall consisting essentially of said crosslinked compositematrix of collagen and soy protein isolate.
 8. The composite nerveconduit of claim 1, wherein said soy protein isolate is high ingenistein.
 9. The composite nerve conduit of claim 1, wherein saidcomposite comprises a weight ratio of collagen and soy protein isolateof from about 20:80 to about 80:20.
 10. The composite nerve conduit ofclaim 1, wherein said collagen is type I collagen.
 11. A method ofpromoting nerve regeneration at a site of neural tissue damage bybridging wounded, severed, or damaged nerve sections in a peripheraland/or central nervous system, said method comprising contacting onedamaged nerve end with a first end of a composite neural conduitaccording to claim 1, and contacting a second end of the neural conduitwith an opposing damaged nerve end.
 12. The method of claim 11, furthercomprising applying an electric current to said site of neural tissuedamage.
 13. The method of claim 12, wherein said electric current isconfigured to generate a unidirectional electric field at said site ofnerve tissue damage from one damaged nerve end to the opposing damagednerve end.
 14. A method of fabricating a composite neural conduit, saidmethod comprising: providing a biopolymer solution comprising collagenand soy protein isolate dissolved or dispersed in a solvent system;applying said biopolymer solution to a negative mold to form the body ofthe conduit, said mold including a plurality of elongated removableinserts for forming one or more hollow elongated internal channelswithin the body, solidifying the biopolymer solution to yield acrosslinked matrix comprising a composite of collagen and soy proteinisolate; and removing said crosslinked matrix from said mold to yield anelongated body comprising the one or more hollow elongated internalchannels extending between respective terminal ends of the body.
 15. Themethod of claim 14, wherein said biopolymer solution consists of saidcollagen and soy protein isolate dissolved or dispersed in said solventsystem.
 16. The method of claim 14, wherein said solidifying thebiopolymer comprises crosslinking collagen and soy protein isolate toyield said crosslinked matrix.
 17. The method of claim 16, wherein saidcrosslinking comprises adding one or more crosslinking agents to thebiopolymer solution.
 18. The method of claim 17, wherein saidcrosslinking agents are selected from the group consisting of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) andN-hydroxysuccinimide (NHS), transglutaminase, glutaraldehyde,sulfonates, genipin, and combinations thereof.
 19. The method of claim17, further comprising washing said crosslinked matrix to removeresidual crosslinker.
 20. The method of claim 14, further comprisinglyophilizing said crosslinked matrix before removing from the mold.